EIA vaccine and diagnostic

ABSTRACT

The invention provides an equine infectious anemia (EIA) vaccine that provides immunity to mammals, especially equines, from infection with equine infectious anemia virus (EIAV) and which allows differentiation between vaccinated and non-vaccinated, but exposed, mammals or equines. Preferably said vaccine encompasses at least one mutation in an EIAV which produces a non-functional gene in the vaccine virus that is always expressed in disease-producing wild-type EIA viruses. Additionally, said EIA vaccine virus cannot cause clinical disease in mammals or spread or shed to other mammals including equines.

RELATED APPLICATION

This application is a divisional of application Ser. No. 09/658,547,filed on Sep. 9, 2000 now U.S. Pat. No. 6,585,978.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to an EIA vaccine, which provides immunity fromdisease and/or infection with EIAV, which vaccine allows diagnosticdifferentiation between vaccinated and non-vaccinated, but exposed ordiseased mammals. More specifically, this invention pertains to avaccine comprising an EIAV wherein an accessory gene has been madenonfunctional and wherein said nonfunctional accessory gene still allowsthe EIAV to replicate in tissue culture.

2. Brief Description of the Prior Art

The equine infectious anemia virus is a member of the lentivirussubfamily of retroviruses and causes persistent infection and chronicdisease in horses worldwide. As such, it is closely related to humanimmunodeficiency virus (HIV), simian immunodeficiency virus (SIV) andfeline immunodeficiency virus (FIV). As with HIV and SIV, disease causedby EIAV is spread by blood transmission. With EIAV, the bloodtransmission most often occurs by biting flies and other insectscarrying virus particles from one horse to another. The first cycle ofdisease (clinical episode or first febrile episode) in an infected horseusually occurs within 42 days after transmission of the virus. Thisfirst cycle is usually characterized by the acute stage of EIA andmanifested by pyrexia, thrombocytopenia, anorexia, depression and highplasma viremia levels. Anemia is not usually detected at this stage.Resolution of this first febrile episode is normally observed after 1 to5 days and occurs concomitantly with a dramatic drop in the amount ofplasma-associated virus. Following the acute stage, some animals mayremain clinically normal, while others go on to experience multiplebouts of illness in which severe anemia may accompany pyrexia,thrombocytopenia, edema, and dramatic weight loss, and death. Nucleotidesequence data has revealed a high mutation rate of this lentivirusgenome during persistent infection (Payne et al, Virology, 1987: 161, p.321–331) incorporated herein by reference. It is generally known thatmultiple isolates from the field demonstrate similar genomic differencesindicating that EIAV, as HIV and FIV, undergoes a continuing mutationprocess within its various hosts. It is generally thought thatneutralizing antibodies aid in the selection of new antigenic virusvariants (mutations) during persistent infections. In infections withEIAV, serologically distinct variants emerge possibly through immuneselection pressure operating on random viral genome mutations. It isproposed that horses that show no further clinical signs of disease havedeveloped a mature immune response that can contain the virus and itsimmunologically-recognized mutants.

The disease is significant because horses that demonstrate exposure toEIAV via testing for antibodies in the blood (Coggins Test or similaranti-p26 antibody detecting test) are either required to be destroyed orstrictly quarantined. Because of the Coggins Test and its broad use inthe world, especially in testing all performance horses that aretransferred into and out of the United States, it is critical thatvaccinated equines be able to be differentiated from infected equines.

The genetic organization of EIAV, as with HIV, SIV and FIV contains onlythree accessory genes (S1, S2 and S3), in addition to the gag, pol andenv genes common to all retroviruses. The S1 open reading frame (ORF)encodes the viral Tat protein, a transcription trans activator that actson the viral long-terminal-repeat (LTR) promoter element to stimulateexpression of all viral genes. The S3 ORF encodes a Rev protein, apost-transcriptional activator that acts by interacting with its targetRNA sequence, named the Rev-responsive element (RRE), to regulate viralstructural gene expression. The S2 gene is located in the pol-envintergenic region immediately following the second exon of Tat andoverlapping the amino terminus of the Env protein (see FIGS. 1, 2 a and2 b). It encodes a 65-amino-acid protein with a calculated molecularmass of 7.2 kDa, which is in good agreement with the size of an in vitrotranslation product. S2 appears to be synthesized in the late phase ofthe viral replication cycle by ribosomal leaky scanning of atricistronic mRNA encoding Tat, S2 protein, and Env, respectively. TheORF coding for the S2 protein of EIAV is highly conserved in allpublished EIAV sequences and contains three potential functional motifs(FIG. 2 a): GLFG (putative nucleoporin motif), PXXP (putative SH3 domainbinding motif) and RRKQETKK (putative nuclear localization sequence).Antibodies to S2 protein can be found in sera from experimentally andnaturally infected horses, indicating that S2 is expressed during EIAVreplication in vivo. These observations suggest that S2 is likely toperform an important role in the virus life cycle. A discussion of thefunction of S2 is found in Li et al (J. Virol., October 1998, p8344–8348), incorporated herein by reference.

A second interesting gene contained within the lentivirus group codesfor dUTPase. This enzyme catalyzes the conversion of dUTP to dUMP andpp₁. The gene encoding the dUTPase has been mapped within the pol genefor EIAV and FIV. The lentivirus dUTPase gene has been designated DU.Studies with DU deletion mutants (ΔDU) of EIAV and FIV show that thisenzyme is not required for replication of the viruses in fetal equinekidney cells or Crandell cells. However, efficient replication of theEIAV or FIV in monocyte/macrophage cells (typical replication host cell)does require DU. The differences indicated have been described in detailin a publication by Lichtenstein et al (J. Virol., May 1995, p2881–2888), incorporated herein by reference.

Envelope proteins (env) are thought to be required for protection fromdisease and, perhaps, protection from infection. By protection fromdisease is meant that a mammal exposed to the virus, does notdemonstrate clinical signs (fever, lethargy, anemia, etc.) but doescarry particles associated with the viral RNA genome (shortened hereinto viral particles) in its blood, said particles being detectable by areverse transcriptase polymerase chain reaction test (RT-PCR). Byprotection from infection is meant that a mammal exposed to the virusdoes not demonstrate clinical signs nor does its blood containRT-PCR-detectable virus particles as described above. The major envelopeproteins of EIAV are gp90 and gp45. These are proposed as the protectiveantigens of EIAV. By the term protective antigens is meant antigens fromEIAV that produce either protection from disease or protection frominfection as indicated above.

It would seem obvious to prepare a vaccine by purifying out the envproteins, especially gp90 and gp45. Indeed, preparation of vaccinescomprising gp90 and gp45 has been attempted with essentially no success.Issel et al (J. Virol. June 1992, p 3398–3408) report that a gp90/gp45vaccine protected ponies from infection caused by homologous EIAV (thesubunits were derived from the same EIAV strain as was used forchallenge), however, these subunits did not protect ponies from eitherdisease or infection when challenged with a heterologous EIAV strain. Infact, the latter produced enhanced disease signs. The subunitenhancement corroborates findings with SIV and FIV subunit vaccines thatappear to enhance disease post challenge. These authors conclude thatperfecting a subunit vaccine for lentiviruses (e.g., HIV, SIV, EIAV andFIV) poses a significant challenge because of the subunit enhancementeffect.

Issel, et al (J. Virol., June 1992, pp 3398–3408) report the preventionof infection by a high-dose whole-virus EIA vaccine. However,vaccination of horses with this vaccine produces horses that are CogginsTest positive (anti-p26 antibody positive) and there is no practicalmethod to demonstrate the difference between vaccinated and infectedequines. Due to the previously-mentioned eradication program in effectin the U.S., a whole-virus vaccine is not feasible.

Since there has been no effective and safe method for immunizing mammalsagainst lentiviral diseases, particularly equines against EIAV and sinceEIAV is such a wide-spread and significant disease world-wide, thereremains a long-felt need to prepare such a vaccine.

SUMMARY OF THE INVENTION

The vaccine of this invention provides the first successful vaccine thateffectively and safely immunizes mammals, especially equids, fromdisease and/or infection caused by EIAV wherein vaccinated mammals canbe differentiated from wild-type EIAV infected mammals.

This invention describes a vaccine for effectively and safely immunizingmammals, especially equids, from disease caused by EIAV, said vaccinecomprising a gene-mutated EIAV wherein said virus lacks the ability toexpress the mutated gene protein in vivo and wherein said lack ofexpression can be used to differentiate vaccinated from non-vaccinatedor infected mammals.

Encompassed within this invention is an EIAV wherein said virus containsa mutation in a gene that allows replication of the virus in vitro suchthat large-scale production can be accomplished.

Also encompassed within this invention is an EIAV wherein said viruscontains a mutation of the S2 gene or portions thereof (ΔS2), a mutationin the DU gene (ΔDU) or a portion thereof, a mutation in a regulatorygene that inhibits expression of the S2 or DU genes or a combination oftypes of said mutations (ΔS2ΔDU). It is expected that further mutationscan be made such that the EIAV in the vaccine contains multiplemutations in multiple genes including the ΔS2, ΔDU or both.

It is within the scope of this invention that a diagnostic test can beused to differentiate vaccinated equines from non-vaccinated and/orinfected equines by measuring the presence or absence of antibodies tothe S2 protein, to the DU protein or to both proteins. Also, a PCR-baseddiagnostic test could be used to detect the presence or absence of theS2 and/or DU genes or gene sequences in the equine and, thus, detectwhether an equine had been infected with EIAV or vaccinated with thecomposition of this invention.

Finally, it is expected that said mutated regions could serve as pointsfor insertion of foreign genes or gene sequences and that said ΔS2 orΔDU or combination thereof with a foreign gene insert could be useful asa vector for vaccination against diseases of mammals other than EIA.Preferably, the insertion would be placed into the ΔDU region.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of replication competent EIAV includingthe location of the accessory genes of EIAV_(UK).

FIG. 2 a is a schematic representation of the EIAV S2 gene and mutantclones derived from EIAV_(UK).

FIG. 2 b is a schematic representation of the Wild-type EIAV S2 genecompared with the EIAV.2M/X (EIAV_(UK)ΔS2) gene.

FIG. 3 a is a circular map of biological proviral clone EIAV_(PR).

FIG. 3 b is a circular map of molecular infectious clone EIAV_(UK).

FIG. 3 c is a circular map of mutant EIAV_(UK)ΔS2.

FIG. 3 d is a circular map of mutant EIAV_(PR)ΔS2.

FIG. 3 e is a circular map of mutant EIAV_(UK)ΔDUΔS2

FIG. 4 are graphs demonstrating the in vitro replication of EIA virusmutant clones.

FIG. 5 is a schematic representation of the DU gene location andconstruction of EIAVΔDU.

DETAILED DESCRIPTION OF THE INVENTION

This invention encompasses a composition for effectively and safelyimmunizing mammals from disease caused by EIAV, said compositioncomprising a gene-mutated EIAV wherein said virus lacks the ability toexpress the mutated gene protein in vivo and wherein said lack ofexpression can be used to differentiate vaccinated from non-vaccinatedor infected mammals. It is contemplated that any gene could be mutatedfrom any EIAV as long as the mutated gene would allow large-scaleproduction of EIAV or EIAV particles. It is further contemplated thatmore than one gene from EIAV could be mutated. It is also understoodthat the composition of the present invention produces a mature immuneresponse capable of protecting equines from disease caused byheterologous as well as homologous EIAV strains.

By gene-mutated is meant that one or more deletions or insertions aremade in a gene of EIAV which makes the gene non-functional and thus,differentiating between the gene-mutated virus and wild-type virus. Thegene mutation can either be produced biologically, by passaging thevirus through cells, cell lines or animals until it becomesnon-infective and a gene-mutated virus is produced, or molecularly(produced by recombinant techniques). By non-functional is meant thatthe gene does not express its protein or product at all or is notexpressing its normal gene product. By a differentiating gene is meantthat the gene product is normally expressed by wild-type EIAV andantibodies to the gene product are found in infected horses but not inhorses vaccinated with the vaccine compositions of the presentinvention. By large-scale production is meant that the gene-mutated EIAVcan be grown or replicated in vitro such that large quantities (e.g., >1liter, preferably greater than 10 liters) can be produced for vaccinemanufacture. Such large-scale production is accomplished if a virus orvirus construct can be produced that is stable, withstands concentrationand/or purification, if necessary, is stable to adjuvants and storage asa vaccine for up to 18 months. By deletion is meant that all or aportion of a gene of EIAV is removed thus causing the gene to becomenon-functional. By insertion is meant that all or part of another geneor a sequence of nucleotides (e.g., a stop codon) is inserted into agene causing it to express a different protein (e.g., one expressed bythe inserted gene) and become non-functional for the normally-expressedprotein or become non-functional by the insertion of a stop codon.Encompassed by this invention is an EIAV wherein said virus contains amutation of the S2 gene or portions thereof (ΔS2), a mutation in the DUgene (ΔDU) or portions thereof, a mutation in a regulatory gene thatcontrols expression of S2 or DU, or a combination of such mutationsaffecting both genes (ΔS2ΔDU). Said mutations would produce anon-functional S2 and/or DU gene. Illustratively, it has beendemonstrated that placing a stop codon into the S2 gene, replacing aminoacid G⁵ produced a non-functional S2 gene. Additionally, it has beendemonstrated that changing the S2 gene's M¹⁶ to T and replacing the G⁵and G¹⁸ with stop codons produced a non-functional S2 gene. Finally, ithas been demonstrated that deletion of the initial 5 nucleotides of S2produced a non-functional S2 gene. Therefore, mutations in the S2 genehave produced EIAV with non-functional S2 genes. The following is anillustration, but non-limiting description of how to produce the abovemutations. Two adjacent fragments were amplified by PCR spanning thewhole S2 gene. One of the two resultant PCR products carried thespecific substitution or deletion mutations incorporated into a PCRprimer. The flanking PCR products were phosphorylated, ligated, and thenused as a template for a second round of PCR with the outer primer pair.The final full-length PCR product was digested with NcoI and Bpu1102I,cloned into EIAV_(UK) previously digested with NcoI and Bpu1102I. Allplasmid clones were sequenced to verify introduced mutations to ensurethe integrity of the PCR-amplified sequence. It is important to notethat the above-identified mutant EIAV clones replicated well in vitro,especially in fetal equine kidney cells (FEK), in equine bloodmonocyte-derived macrophage cells (MDM) or an equine dermal cell line(ED). Therefore, these gene-mutated EIAV clones can be produce inlarge-scale and have been used to prepare a vaccine for safe andeffective immunization of horses.

As would be recognized, mutations comprising deletions could be madesuch that the EIAV contained multiple deletions in genes including theS2 (ΔS2), DU (ΔDU) or both. A gene-mutated EIAV comprising a deletion inthe DU (ΔDU) gene was prepared by deleting a StyI restriction fragmentcontaining 80% of the DU coding sequence, including four of the fiveconserved amino acid motifs, from the proviral clone designatedPV19-2-6A (described by Lichtenstein et al, J. Virol. May 1995, p.2881–2888 and incorporated herein by reference). It has beendemonstrated that the above-described deletion in the DU gene does notreduce the ability of this gene-mutated EIAV to replicate in eitherfetal equine kidney cells (FEK) or in an equine dermal cell line (ED)both considered to be in vitro growth. Therefore, it has beendemonstrated that this gene-mutated EIAV can be produced in large-scaleand vaccine production is possible.

In accord with the invention, it has been found that the S2 antibodiescan be detected in horses with EIAV infections by using immunoassayscomprising recombinant S2 protein or synthetic S2 peptides as thecapture antigen. Additionally, it has been determined that the presenceof the type of virus found in a mammal can be differentiated between thevaccine virus and the wild-type virus by use of gene probes (PCR-based).It has also been determined that the S2 gene of EIAV is not required forin vitro replication in a variety of equine cells including but notlimited to festal equine kidney cells (FEK), equine dermal cell lines(ED) or cultured equine monocytes/macrophages. It has further beendetermined that the S2 deletion mutant replicates in vivo only at verylow levels as compared with the wild-type EIAV (Li, et al, January 2000,J. Virol. pp 573–579), incorporated herein by reference. By low levelsis meant that the virus produces less than 1×10⁵ EIAV particles (asdetected by PCR) in vivo, preferably less than 1×10⁴. Further, it hasbeen determined that the S2 protein is not a component of purified EIAVparticles and that horses immunized with purified EIAV particles do notproduce serum antibodies reactive with in vitro synthesized S2 proteinor peptides. Therefore, even horses vaccinated with purified EIAVparticles can be differentiated from wild-type infected horses. Theseresults indicate that the presence of S2 specific antibody can be usedto identify EIAV-infected horses and to distinguish infected horses fromthose that have been vaccinated with an inactivated whole virus or anattenuated vaccine in which the S2 gene is mutated so as to make itnon-functional. Therefore, it is within the scope of this invention thata diagnostic test can be used to differentiate vaccinated equines fromnon-vaccinated and/or infected equines by measuring the presence orabsence of antibodies to the S2 protein, to the DU protein or to bothproteins. Such differentiation can be measured by developing animmunoassay, an antibody-detecting assay (e.g., indirect fluorescentantibody, immunodiffusion, agar diffusion, electrophoresis) or aPCR-based assay known to the art. An example of an immunoassay is anenzyme linked immunosorbent assay (ELISA) that detects and/orquantitates antibodies to specific proteins in serum, blood or tissues.ELISA technology could also be used to detect the presence or absence ofvirus-associated antigens in the blood, serum or tissues. By virusassociated antigens is meant the presence or absence of a geneexpression product such as the S2 or DU proteins in the case of the S2or DU genes, respectively. Additionally, PCR-based assays have been usedto measure the presence or absence of genes or gene sequences in theblood, serum or tissues of an equine, thus indicating that a horse hadbeen infected or vaccinated, as the case may be. For this particularembodiment, an ELISA would detect the presence of antibodies to the S2or DU proteins. If antibodies were present in horses that were tested itwould indicate that the horse had been infected with EIAV. Horses thathad been vaccinated with a gene-mutated EIAV construct containing anon-functional S2 gene would not contain S2 antibodies in their serum.Horses that had been vaccinated with a gene-mutated EIAV constructcontaining a non-functional DU gene would not contain DU antibodies intheir serum. Thus, vaccinated horses could be differentiated frominfected horses. The PCR-based assays would be used to detect thepresence or absence of gene sequences within the horse. For instance, ifa horse had been infected with a wild-type EIAV, it would contain thegene sequence for wild-type S2 or DU. However, equines immunized withvaccines comprising a gene-mutated EIAV, particularly one wherein the S2or DU genes comprised deletions or specific mutations would not containthe gene sequence for wild-type S2 or DU gene products.

As would be recognized from this invention, said mutated (deleted) generegions could serve as potential points for insertion of foreign genesand that said ΔS2 or ΔDU or a combination thereof, preferably within theΔDU, with a foreign gene insert could be useful as a vector forvaccination against diseases of mammals other than EIA and could serveto protect mammals from a second type of viral, bacterial or parasiticdisease. For instance, it would be highly advantageous to incorporate agene for another important equine disease (e.g., equine influenza,equine herpes virus types 1, 2, or 4, Streptococcus equi, Rhodococcusequi) into the gene-mutated EIAV. When such a vaccine is used tovaccinate horses, the horse would not only be protected from diseasecaused by EIAV but also from disease caused by the other equine diseaseorganism.

Vaccines of the present invention have been either inactivated oradministered live. Inactivated vaccines of the present inventioncomprise treatment of the live virus, attenuated virus, purified virusparticles or whole virus particles with agents that inactivate the virussuch that it cannot replicate in vitro or in vivo. Such agents areselected from the group consisting of formalin, formaldehyde,beta-propriolactone, binary ethyleneimine, ethyleneimine, merthiolate,thimerosal, psoralen and combinations thereof. These agents can be usedat concentrations varying from 1 part per billion to 0.5%, depending onthe agent. For instance, thimerosal would be used at a concentration ofbetween 1 part per 1,000 and 1 part per billion, preferably between 1part per 5,000 and 1 part per 100,000. Formalin would be used at aconcentration between 0.00001% and 0.5%, preferably between 0.0001% and0.1%. Ethyleneimine would be used at a concentration between 0.00001Mand 0.1M, preferably between 0.0001M and 0.01M. Beta-propiolactone wouldbe used at a concentration similar to that used for ethylenimine.

Vaccines of the present invention may also include adjuvants in order toenhance the immune response. Adjuvants are chemical agents or extractsof microorganisms that induce an enhanced immune response. Whenaccompanied by an antigen, they enhance the immune response produced bythe antigen. In the case of EIAV particles, EIAV purified virusparticles, EIAV constructs, attenuated EIAV, EIAV (whole virus) or EIAVsubunits, adjuvants may be added to enhance the immune response to thevaccine composition to provide improved protection. It is recognizedthat adjuvants would be used according to the present invention atconcentrations varying from 0.1% to 50% v/v, preferably from 1% to 20%.

Although any adjuvant will enhance the immune response and can be usedwith the vaccine compositions of the present invention, it is within theteaching of the present invention that adjuvants selected from the groupconsisting of polymer-based, oil-based, block copolymer-based, aluminumsalt based, organism-based, lipid-based and aqueous-based, surfactantsare preferred. Non-limiting examples of surfactants useful as adjuvantsinclude hexadecylamine, octadecylamine, lysolecithin,demethyldioactadecyl ammonium bromide, N,N-dioctadecyl-N′-N-bis(2-hydroxyethylpropane diamine), methoxyhexa-decyl-glycerol and pluronicpolyols and saponin, Quil A. Non-limiting examples of polyanions orpolycations include pyran, diethylaminoethyl (DEAE) dextran, dextransulfate, polybrene, poly IC, polyacrylic acid, carbopol, ethylene maleicacid, aluminum hydroxide, and aluminum phosphate. Non-limiting examplesof peptide adjuvants include muramyl dipeptide, dimethylglycine andtuftsin. Non-limiting examples of other types of adjuvants include oilemulsions, immunomodulators (interleukin-1, interleukin-2 andinterferons) or combinations of any of the foregoing adjuvants. A numberof acrylic acid polymers and copolymers of acrylic acid and methacrylicacid and styrene have adjuvant activity. Polyvinyl Chemical Industries(Wilmington, Mass.) provides such polymers under the trade-nameNEOCRYL®, BEOCRYL A640, an aqueous acrylic copolymer with styrene. Otheruseful NEOCRYL products are 520 and 625, and NEOREZ 966. Ethylene maleicacid, produced from ethylene maleic anhydride is a preferred adjuvant.In order to produce ethylene maleic acid, EMA 31 or EMA 91 (MonsantoCo., St. Louis, Mo.) is prepared in an aqueous solution at aconcentration between 0.1 and 10% (w/v), preferably between 0.5 and 5%(w/v). It is used in product at a concentration of 1 to 50% (v/v). Morepreferably, Carbopol is used as an adjuvant alone or in combination withtweens, spans and oils. Representatives of this type of adjuvant areHAVLOGEN® and SPUR®. These adjuvants are prepared by mixing Carbopol934P at a concentration between 0.5 and 10% (w/v), preferably between 1and 5% (w/v), more preferably between 2.0 and 4% (w/v). Added to theCarbopol can be detergents such as Tween 80 and Span 20, and an oil forproducing an emulsion. The oils can be cottonseed, peanut, mineral, orany other type known to be safe for use in animals. The concentrationsof the oil ranges from 0.000001% to 10% (v/v), preferably from 0.00001%to 5% (v/v), more preferably from 0.0001% to 1% (v/v). Othercommercially-available adjuvants useful for this vaccine include but arenot limited to POLYGEN™, a polymer-based low molecular weight,non-particulate copolymer which can form cross-linkages in solution tobecome a high molecular weight gel (MVP Laboratories, Inc., Ralston,Nebr.) or EMULSIGEN™ or EMULSIGEN™ PLUS, both oil-in-water adjuvantsprovided by MVP Laboratories, Inc. Organism-based adjuvants are thoseutilizing whole microorganisms or extracts of microorganisms, such asMuramyl Dipeptide, RIBI®, whole Parapox viruses or extracts thereof(also known as Baypamun) and Corynebacterium acne extracts. Lipid-basedadjuvants include but are not limited to BAY R1005, liposomes andISCOMS. The most preferred adjuvants of the present invention includeHAVLOGEN®, POLYGEN™, BAY R1005, Baypamun and ethylene maleic acid-based.Often, two or more adjuvants can be used to formulate with the EIAVconstructs of this invention.

In order to better understand the following Examples, the wild-type EIAVis referred to as the Wyoming isolate or EIAVwyo. This virus is termed aprimary isolate and it replicates only in equine monocyte-macrophagecell cultures in which the virus is cytopathic for the infected cells by7–10 days post infection. Thus, EIAVwyo can be produced only inshort-term macrophage cultures to obtain infectious virus in cellsupernatants or in experimentally infected horses to obtain infectiousplasma (Malmquist et al. 1973, Arch. Virol. 42, p 361–370). Eithersource of the primary isolate EIAVwyo can be used to experimentallyinfect equids and produce classical EIA disease. To obtain acell-adapted strain of EIAVwyo that is able to replicate in other celltypes, the primary EIAVwyo isolate was serially passaged in equine cellsto produce a stock of EIAV virus that could be grown on variousfibroblastic cells (Malmquist et al 1973, Arch Virol. 42, p 361–370;Parekh et al. 1980 Virology 107:520–525). The cell-adapted EIAVwyo wasthen grown in fetal equine kidney cell cultures to produce largeramounts of virus and thus used to prepare stocks of the cell-adaptedvirus designated EIAV_(PR) (Montelaro et al. 1982 J. Virology42:1029–1038). Inoculation of ponies with the avirulent EIAV_(PR)results in 100% infection but does not produce EIA disease, confirmingthe attenuated avirulent nature of the EIAV_(PR) strain (Orrego et al.,1982 Am. J. Vet. Res. 43:1556–1560). To obtain a reference strain ofEIAV that can be grown in fibroblastic cells and produce disease inexperimentally-infected equids, the EIAV_(PR) strain was seriallypassaged in ponies and isolated in the context of infectious plasmaafter the third serial passage (Orrego et al. 1982 Am. J. Vet. Res.43:1556–1560). The in vivo serial passage restored virulence to theEIAV, but did not cause it to lose its ability to replicate in cellsother than equine macrophages. This virus stock in infectious plasma wasdesignated as host-adapted EIAVwyo. Inoculation of ponies withhost-adapted EIAVwyo induced 100% infection and clinical EIA disease(Payne et al. 1987 Virology 161:321–333). In a subsequent set ofexperiments, a host-adapted EIAVwyo was grown in fetal equine cellculture in the presence of neutralizing immune serum from a pony togenerate antigenic neutralization escape mutants by antibody selectionthat were then biologically cloned to obtain a more homogeneous genomicpopulation (Rwambo et al. 1990 Arch. Virol. 111:275–280). Subsequentstocks of this biologically cloned reference virus produced in fetalequine kidney cell culture were termed EIAV_(PV) to indicate “ponyvirulent”. Infection of ponies with the biologically cloned EIAV_(PV)results in 100% infection and disease (Hammond et al. 1997 J Virology71:3840–3852). Since lentiviruses like EIAV exist in nature as complexgenomic mixtures termed quasispecies, primary isolates (EIAVwyo) andbiological clones (EIAV_(PV)) contain a variety of genomic species. Toobtain genetically homogenous forms of EIAV, infectious molecular cloneswere derived from the avirulent EIAV_(PR) (e.g., EIAV 19-2) (Payne et al1994 J. Gen. Virol. 75:425–429) and pathogenic EIAV_(PV) (Cook et al.1998 J. Virology 72:1383–1393) reference stocks by standard molecularbiology cloning procedures. Inoculation of ponies with infectious virusstocks produced from chimeras with EIAV_(PR) and EIAV_(PV) sequences(e.g., EIAV_(UK)) were shown to produce disease inexperimentally-infected horses. The infectious molecular clone EIAV_(UK)was the first reported pathogenic molecular clone.

FIG. 3 b displays the circular map of this infectious molecular clone,EIAV_(UK) In order to provide further information for the followingexamples, FIG. 3 c displays the circular map of EIAV_(UK)ΔS2, FIG. 3 ddisplays the circular map of EIAV_(PR) ΔS2, and FIG. 3 e displays thecircular map of EIAV_(UK)ΔDUΔS2.

The invention is further illustrated but is not intended to be limitedby the following examples in which all parts and percentages are byweight unless otherwise specified.

EXAMPLE 1

Several different gene-mutated EIAV constructs were prepared accordingto the methods of Li et al (J. Virol., October 1998, pp 8334–8348) whichare incorporated herein by reference. The basic S2 gene mutations weredesigned so as not to disrupt the second exon of Tat 10 base pairs (bp)upstream from the S2 initiation sequence, the envelope initiator codonjust 23 bp downstream from the S2 start codon sequence, or the putativeRev-response element (RRE) sequences that have been mapped to both the5′ and 3′ ends of the env gene. A panel of clones with substitutionsthat introduce one or more premature stop codons (EIAV.2M/X and EIAV.G5/s) or with a deletion of the first 5 nucleotides of the S2 gene to shiftthe S2 ORF (EIAVΔS2) were produced. These are schematically diagramed inFIGS. 2 a and 2 b. The EIAV proviral DNA is shown at the top; thecomplete deduced amino acid sequence of the putative S2 protein is shownin single letter amino acid code at the bottom. Stop codons (indicatedby arrows) were introduced into various positions in the EIAV S2 gene togenerate the specific mutant virus strains. As would be recognized, allof the constructs would be considered to be non-functional for S2 andwill be referred to herein as ΔS2.

S2 mutant constructs were generated using the PCR-Ligation-PCR (PLP)strategy as previously described (Puffer, et. al., 1997 and Li, et. al.,1998). EIAV_(UK) plasmid DNA was used as the template to perform all PCRreactions for generating S2 mutations except for EIAV_(UK).2M/X.

EIAV.G5/ s was generated using EIAV_(UK) as the template by PCR with Pfupolymerase (Stratagene) by using mutagenic downstream primer mspe3–5′(SEQ ID NO: 1) with upstream primer s2pst (SEQ ID NO: 2). A secondflanking fragment was amplified using mutagenic upstream primersmspe5′-3′ (SEQ ID NO: 3) and s2sph (SEQ ID NO: 4).

EIAV_(UK)ΔS2 was similarly generated using EIAV_(UK) as the template byPCR with Pfu polymerase (Stratagene) by using downstream primerS2min/35rev (SEQ ID NO: 5) and upstream primer s2pst (SEQ ID NO: 2). Asecond flanking fragment was amplified using mutagenic upstream primerS2min/53for (SEQ ID NO: 6) and s2sph (SEQ ID NO: 4).

Each of these corresponding two adjacent PCR fragments were gelpurified, phosphorylated using T4 polynucleotide kinase (Gibco BRL), andligated by using T4 DNA ligase (Gibco BRL). After inactivation at 65° C.for 15 minutes, the ligation reaction was used for a subsequentamplification using upstream primer s2pst (SEQ ID NO: 2) and downstreamprimer s2sph (SEQ ID No: 4). This product was gel purified, digestedwith NcoI and Bpu1102I, and then ligated into the NcoI and Bpyu1102Isites of EIAV_(UK).

EIAV_(UK).2M/X, which has its sequence compared with that of EIAV_(UK)in FIG. 2 b, was generated using the EIAV_(UK)G5/ s plasmid DNA as atemplate with downstream primer 2M35/RE (SEQ ID NO: 7) and upstreamprimer s2pst (SEQ ID NO: 2). A second flanking fragment was amplifiedusing mutagenic upstream primer 2M53/ For (SEQ ID NO: 8) and downstreamprimer s2sph (SEQ ID NO: 4). The final cloning procedure was asdescribed above.

For simplification and because all of the EIAV constructs described arenon-functional for S2 as demonstrated in tissue culture growth studies(as described in EXAMPLE 2), these EIAV constructs have beenredesignated EIAV_(UK)ΔS2.

Standard PCR conditions used for the above-described reactions included,one cycle of denaturation at 95° C. for 5 min., followed by 35 cycles ofdenaturation at 95° C. for 30 seconds, 60° C. for 30 seconds and 72° C.for 30 seconds. The PCR reactions were set up using the followingcomponents:

-   -   10 μL 10× NEB Thermophilic buffer    -   1.0 L μ10 mM deoxynucleotide triphosphates dNTPs    -   1.0 μM forward primer (upstream primer)    -   1.0 μM reverse primer (downstream primer)    -   10 ng template DNA    -   x μL double distilled water (ddH₂O) (q.s. to 100 μL volume)

A 10 μL aliquot was run on an 1.0% agarose gel to make sure the correctsize product was amplified. The PCR products were then gel isolated andpurified with a Qiaex II gel extraction Kit (150)(Qiagen, Cat. # 20021).The Qiaex II protocol is presented below:

1. Cut band from gel and place in a 1.5 mL eppendorf tube.

2. Estimate the volume of agarose gel slice, add 3 volumes of bufferQ×1, if the fragment is <4 kb, and an additional 2 volumes of ddH₂O ifthe fragment is >4 kb.

3. Vortex the Qiaex II beads and add 10 μL to the agarose slicesuspension.

4. Mix well, incubate at 50° C. for 5–10 minutes, mixing the tubeseveral times during the incubation period.

5. Centrifuge the sample for 30 seconds and carefully remove thesupernatant with a pipette followed by washing the pellet once with 500μL of buffer Q×1.

6. Wash the pellet twice with 500 μL of buffer PE, and air dry pellet15–30 minutes at room temperature.

7. Resuspend the pellet in 20 μL of ddH₂O, incubate at 55° C. for 10min., spin at full speed for 30 seconds.

8. Pull off supernatant and save to a clean eppendorf tube. Measure theOD at 260 nm for the concentration of the recovered fragment on anagarose gel.

9. Add ddH2O as needed to resuspend the pellet.

The two adjacent PCR fragments were individually phosphorylated in thefollowing reaction mixture by using T4 polynucleotide kinase (NEB) priorto ligation. The phosphorylation reaction was set up as follows:

-   -   2.0 μL 10×T4 polynucleotide kinase (PNK) buffer (NEB)    -   2.0 μL 10 mM ATP (NEB)    -   1.0 μL T4 PNK (NEB)    -   15 μL gel purified DNA of each of these two adjacent PCR        fragments

The reaction was incubated at 37° C. for 1 hour. Following inactivationat 65° C. for 10 min. the adjacently phosphorylated PCR fragments werethen ligated together by using T4 DNA ligase (NEB) under the followingconditions:

-   -   1.0 μL 10×T4 DNA ligase buffer (NEB)    -   X μL (50–100 ng) of each of two adjacent PCR fragments    -   1.0 μL T4 DNA ligase (NEB)    -   X μL ddH2O (q.s. to 10 μL total volume)

After overnight incubation at 16° C. the ligation reaction product wasused in a second round PCR reaction to amplify the full-length PCRfragment spanning these two adjacent PCR products. The second round PCRreaction was performed as previously described (see below) with theexception that only upstream primer s2pst (SEQ ID NO: 2) and downstreamprimer s2sph (SEQ ID NO: 4) were used. Again, a 10 μL aliquot was run onan agarose gel to make sure the correct product was amplified. Thefull-length PCR fragments were then gel isolated and purified using theQiaex II kit (see above). The purified full-length PCR fragment,together with EIAV_(UK), were then cut with NcoI (Gibco BRL) and Bpu1102I (Gibco BRL) under the following conditions:

-   -   2.0 μL 10× React2 buffer (Gibco BRL)    -   1.0 μL NcoI (Gibco BRL)    -   1.0 μL Bpu1 102I    -   X μL full length PCR product (1.0 μg) or EIAV_(UK) (500 ng)    -   X μL ddH₂O (q.s. to 20 μL total volume)

The above restriction enzyme digestion mixture was incubated at 37° C.for 2 hours. Digested DNA fragments from the full-length PCR product andthe EIAV_(UK) plasmid were individually gel isolated and purified usinga Qiaex II kit as described above. The digested vector EIAV_(UK) andfull length PCR fragment were ligated using T4 DNA ligase using thefollowing procedure:

-   -   1.0 μL 10×T4 DNA ligase buffer (NEB)    -   X μL (25–50 ng) digested EIAV_(UK)    -   X μL (200–400 ng) digested full length PCR fragment    -   1.0 μL T4 DNA ligase (NEB)    -   X μL ddH2O (q.s. to 10 μL total volume)

The ligation reaction was incubated at 16° C. overnight and the ligatedproducts were transformed into Escherichia coli DH5α (Gibco BRL) by heatshock as described below:

-   -   1. Thaw 100 μL of DH5α competent cells and incubate on ice    -   2. Add 1 μL of ligation mixture to cells, mix gently, and        incubate on ice for 30 minutes    -   3. Heat pulse the tube in a 42° C. bath for 45 seconds and        incubate on ice for 2 minutes.    -   4. Add 0.9 mL SOC broth (2% bactotryptone, 0.5% yeast extract,        10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄ and 20 mM        glucose, pH 7.0) and incubate the tubes at 37° C. for 1 hour        while shaking at 222 rpm.    -   5. Plate 150 μL of the transformation mixture onto LB-ampicillin        (100 μg/mL) plates and incubate at 37° C. overnight.

The proviral clones (EIAV_(UK) 0.2M/X, EIAV_(UK) G5/s and EIAV_(UK)ΔS2)were then screened by sutomatically sequencing using a Taq Dye DeoxyTerminator Cycle Sequencer Kit (Applied Biosystems) individually usingan internal sense primer S40 (SEQ ID NO: 9) and an internal antisenseprimer S15 (SEQ ID NO: 10). Following the verification for the mutationsin the S2 gene by sequencing, the proviral DNA clones were used forvarious future studies.

The generation of EIAV_(UK)ΔDUΔS2 was based on the modification of thepreviously studied EIAV_(PR)ΔDU virus in which thedeoxyuridinetriphosphatase (dUTPase or DU) gene segment was deleted byremoving a 330-bp StyI restriction fragment (Lichtenstein, et al.,1995). EIAV_(UK)ΔDUΔS2 was generated by subcloning into the full-lengthEIAV_(UK)ΔS2 proviral backbone of a SstI-NcoI fragment of EIAV_(PR)ΔDU,which contained a 330-bp deletion in the DU gene. EIAV_(PR)ΔS2 wascreated by subcloning into the full-length EIAV_(PR) proviral backboneof a NcoI-BpuI102I fragment of EIAV_(UK)ΔS2, which contained a S2 genemutation. All of the various constructs discussed above contain anon-functional S2 gene and could be used in vaccines for immunizinghorses against diseases caused by EIAV. The constructs are compared withthe wild-type EIAV in FIGS. 1 and 2. FIGS. 3 d and 3 e represent thecircular maps of EIAV_(PR)ΔS2 and EIAV_(UK)ΔDUΔS2.

It is expected that each of the gene-mutated EIAV constructs can be usedto prepare either live attenuated or inactivated vaccines for safe andeffective immunization of horses from disease caused by EIAV and can beused to differentiate vaccinated horses from infected horses. Asindicated previously, it is recognized that inactivation would beproduced by adding an appropriate amount of any of the inactivatingagents listed previously or others known in the art to be acceptable tolentiviruses. An appropriate amount means the lowest concentration ofinactivating agent necessary to inactivate all of the virus particleswithout damaging the protective antigens (immunogens).

EXAMPLE 2

In order to demonstrate that the gene-mutated EIAV constructs fromExample 1 could replicate in large-scale, a tissue culture growth studywas conducted. One microgram of proviral clone DNA from each of theconstructs was used to transfect an ED cell line. The ED cell line (ATCCCRL 6288) was grown in 6 well tissue culture plates seeded with between2 and 4×10⁵ ED cells per well in 2 mL of the complete growth MinimumEssential Media with Earles salts (EMEM) plus 10% fetal calf serum, 100units/mL of penicillin, 100 μg/mL of streptomycin (Gibco BRL 15140–122)and 2 mm L-glutamine (Gibco BRL 25030-081). The plates were incubated at37° C. in a CO₂ incubator approximately 16 to 24 hours until the cellswere between 50 and 80% confluent. For each transfection, 1 μg of DNAwas diluted into 100 μL of OPTI-MEM I Reduced Serum Medium (Gibco BRL18324-012) and 10 μL of Lipofectamine reagent (Gibco BRL 18324-012) wasadded to 100 μL of OPTI-MEM I Reduced Serum Medium (OPTI-MEM RSM). Thetwo solutions were mixed gently and incubated at room temperature for 30minutes to allow the DNA-liposome complexes to form. During this time,the ED cell cultures were rinsed once with 2 mL of OPTIMEM I RSM. Foreach transfection, 0.8 mL of OPTI-MEM I RSM was added to the tubecontaining the DNA-liposome complexes, the tube was mixed gently and thecontents were overlayed onto the rinsed cells. No antibiotics were addedduring transfection. The DNA-liposome/tissue cultures were incubated for5 hours at 37° C. in a CO₂ incubator. Following incubation, 1 mL ofcomplete growth MEM containing twice the normal concentration of serumwas added to the cell culture without removing the transfection mixture.Twenty four hours following the start of transfection the medium wasreplaced with fresh complete growth medium (EMEM). Starting at 48 to 72hours post transfection, aliquots of the tissue culture supernatantswere taken at periodic intervals and analyzed by using a standardreverse transcriptase (RT) assay as a measure of virus production.Supernatants resulting in RT activity were titrated in an infectivityassay based on cell-ELISA readings as described by Lichtenstein et al,1995. After titer determination, aliquots of each of the virus constructstocks were frozen at −80° C. for further evaluation and use. All of theconstructs replicated well in both ED cells and in MDM cells producingRT levels of at least 10,000 CPM/10 μL which was the normal level of RTactivity observed in wild-type EIAV_(UK) (See FIG. 4). Further passagingof the transfected cells in larger vessels was accomplished by use ofthe same techniques as described and serves as the basis for indicatingthat the constructs prepared in Example 1 could be produced inlarge-scale and, therefore, could be used to prepare vaccines.

The tissue culture grown virus construct stocks were molecularlycharacterized by extracting viral RNA and conducting RT-PCR analyses ofthe DU and S2 genes using 20% glycerol cushion purified virus constructparticles. These sequence analyses confirmed the DU and/or S2 genemutation in their corresponding virus constructs. The RT-PCR techniquewas also employed to identify recombinant virus construct stocks.Wild-type EIAV_(UK) generated a RT-PCR product of 592 base pairs (bp).In contrast, virus constructs containing the DU deletion(EIAV_(UK)ΔDUΔS2) resulted in a RT-PCR fragment of 262 bp. S2 genemutant virus constructs identified as EIAV_(UK)ΔDUΔS2, EIAV_(UK)ΔS2 andEIAV_(PR)ΔS2 were also analyzed by the RT-PCR technique. While creatingthe S2 mutation, a SpeI restriction digestion site was created. RT-PCRand restriction digestion analyses of each of EIAV_(UK)ΔS2,EIAV_(UK)ΔDUΔS2, EIAV_(PR)ΔS2 and EIAV_(UK) virus stocks demonstratedthat EIAV_(UK) wild-type virus generated a 539 bp RT-PCR fragment thatwas resistant to digestion by SpeI. Each of the above-listed S2 virusconstructs was susceptible to digestion by SpeI, resulting in cleavageof the 539 bp RT-PCR product into 347 and 192 bp fragments.

EXAMPLE 3

In order to prove that the constructs prepared and grown in large-scalein the previous examples could protect either ponies or horses fromdisease produced by EIAV, a vaccine was prepared using proviral cloneEIAV_(UK)ΔS2. The EIAV_(UK)ΔS2 virus construct was grown in primaryfetal equine kidney cells (FEK), filtered through a 0.45 μ filter andfrozen in aliquots at −80° C. The titers of these virus construct stockswere 10⁶ infectious center doses (ICD) per mL, as measured by using anEIAV infectious center assay in FEK cells (Lichtenstein, et al, 1995),incorporated herein by reference.

For these studies, the EIAV_(UK)ΔS2 could have been inactivated,preferably, by using agents such as formalin or binary ethyleneimine.Additionally, the virus construct could have been adjuvanted with any ofseveral adjuvants, preferably with a Carbopol-based, polymer-based orlipid-based adjuvant. However, for this experiment, the EIAV_(UK)ΔS2 wasused without inactivation or adjuvanting so as to determine whether itwould replicate in vivo as well as it replicated in vitro. Thus, thisexample describes the use of an attenuated live vaccine comprisingEIAV_(UK)ΔS2.

The EIAV_(UK)ΔS2 was tested for its ability to protect equines (poniesin this experiment) against an intravenous challenge with pathogenicEIAV_(PV), a heterologous EIAV. The results of thisvaccination/challenge study are shown in Table 1. Each of three ponieswas vaccinated once with 1.0 mL of the undiluted EIAV_(UK)ΔS2 virusconstruct stock. Six months after vaccination all 3 vaccinated ponieswere challenged intravenously with 300 median equine infectious doses(MEID) of pathogenic EIAV_(pv). All ponies were clinically monitored andmaintained in isolation as described by Hammond, et al. (Virology, 1999,vol: 254, p 37–49). Rectal temperatures and clinical status wererecorded daily. Samples of serum, plasma and whole blood were collectedfrom each pony at predetermined intervals. Plasma samples were stored at−80° C. until further processed for semi-quantitative viral RNA analysesor identification of the presence of wild-type challenge virus, andserum samples were stored similarly until testing for quantitative andqualitative serological assays could be performed. Whole blood sampleswere appropriately fractionated for enumeration of platelets orexperimentation with PBMCs. Results are shown in Table 1.

During the course of the 6-month immunization, no clinical signs wereobserved in the vaccinated ponies. This indicates that EIAV_(UK)ΔS2 isavirulent for ponies. To assess virus replication following vaccination,the level of viral RNA in plasma was determined by using asemi-quantitative RT-PCR assay (Li et al, J. Virol, January 2000, p.573–579). EIAV RNA was detected in the plasma of all immunized animalson day 6 after vaccination and unpredictable viremia episodes wereobserved throughout the course of vaccination. However, the plasma viralRNA levels observed in the vaccinated ponies were 10–6000 fold lowerthan the levels measured in ponies previously infected with the parentalEIAV_(UK) virus over a six month observation period. This findingindicates that the EIAV_(UK)ΔS2 virus construct is highly attenuated dueto the absence of the S2 gene and therefore, the vaccine was safe inequids even in live form.

At 6 months post vaccination, the ponies were challenged intravenouslywith 300 median equid infectious doses of pathogenic EIAV_(PV).Following challenge of non-vaccinated ponies, clinical signs of EIA arenormally apparent in about 16–19 days. Concurrent with the initialEIA-related fever is a rapid decline in quantity of plateletscirculating in the blood. In some cases, control ponies recrudesce withmore severe clinical manifestations of high fever (>103° F.) andextremely low blood platelet counts of <105,000/ μL of blood. Followingchallenge in these ponies, clinical signs of EIA were not evident at allthroughout the 3 months observation period indicating that these ponieswere successfully protected from disease.

In order to determine whether the ponies were protected from infection(sterile protection), genetic analyses of viral RNA present in theplasma was performed by a nested RT-PCR technique used in combinationwith differential restriction digestion of RT-PCR product for thedifferentiation of vaccine strain EIAV_(UK)ΔS2 and wild-type challengestrain EIAV_(PV). All of the vaccinated/challenged ponies demonstratedonly the presence of the attenuated EIAV_(UK)ΔS2 virus construct.Therefore, all (100%) were protected from infection by wild-type EIAV.

TABLE 1 Summary of Results of Pony Vaccination/Challenge Study FebrileAbnormal Blood PCR Detection Pony Episode Count Post Protection ofChallenge Protection Group No. Post Challenge Challenge From Disease^(a)strain EIAV_(PR) From Infection^(b) EIAV_(UK)ΔS2 94-11 NONE NONE YESNegative YES 676 NONE NONE YES Negative YES 674 NONE NONE YES NegativeYES ^(a)Animals protected from clinical disease did not demonstrate anyprogression to clinical disease including temperature and platelet count^(b)Animals protected from infection did not demonstrate any level ofexpression of wild-type challenge virus at the plasma samples ofvaccinated horses by a semi-quantitative RT-PCR (Li et al, J. Virol,Jan. 2000)

EXAMPLE 4

A vaccination/challenge study similar to the described in Example 3 wasconducted with horses using the multiple low dose challenge. This studywas conducted in order to demonstrate equivalency between vaccination ofponies or horses as well as demonstration that the multiple low dose EIAequine challenge can serve as a successful model for EIAV infection. Themethod of Example 2 was used for growth of the EIAV_(UK)ΔS2 construct.Six horses were vaccinated with 1.0 mL of the virus construct. One horsewas left unvaccinated to serve as a Control horse. In this study, horseswere challenged using the multiple low dose application Ser. No.09/659,030, incoporated herein by reference. In the multiple low doseEIA equine challenge, each horse received three intravenous inoculationsof 10 median horse infective doses (MHID) of EIAV_(pv) at two-dayintervals. After challenge, the horses were monitored for clinical signsof EJA for about 3 months post challenge. Results of the horse challengeare shown in Table 2.

TABLE 2 Summary of Results of Horse Vaccination/Challenge Study AbnormalFebrile Blood Platelet PCR Detection Horse Episode Count Post Protectionfrom of Challenge Protection Group No. Post Challenge Challenge Diseasestrain EIAV_(PR) From Infection EIAV_(UK)ΔS2 60 NONE NONE Yes NegativeYES 971 NONE NONE Yes Negative YES 615 NONE NONE Yes Negative YES 9791NONE NONE Yes Negative YES 9809 NONE NONE Yes Negative YES 9812 NONENONE Yes Negative YES CONTROL 880 YES YES No Positive NO

These data indicate that a vaccine that protects from both disease andinfection in ponies and/or horses produced by EIAV can be prepared fromEIAV_(UK)ΔS2. Equines vaccinated with the attenuated EIAV_(UK)ΔS2construct can be differentiated from infected equines based on the lackof antibody to the S2 protein in vaccinated animals. Such lack ofantibody can be determined by any immunological assay known to the artthat would demonstrate the presence of S2 antibodies in the blood orserum of infected ponies or horses and the lack of such antibodies invaccinated ponies or horses. Alternatively, a PCR-based assay known tothe art, could be used to detect the presence of the S2 gene sequence ininfected horses as compared to the lack of this gene sequence invaccinated horses. The horse experiment demonstrates that the multiplelow dose EIA equine challenge model is effective in both reproducing EIAand in demonstrating that horses can be protected from by a vaccineprepared according to the present invention.

EXAMPLE 5

Live attenuated vaccines were also prepared from the EIAV constructsdesignated EIAV_(UK)ΔDUΔS2 and EIAV_(PR)ΔS2 according to the methodsdescribed in Example 3. Two groups of horses were each inoculatedintramuscularly two times (at monthly intervals) with the respectiveattenuated vaccine. Each vaccine contained approximately 10⁵infectious-center doses (ICD) in a 1.0 mL dose. Inoculated horses weremonitored daily for any clinical signs of EIA post vaccination. Bloodsamples were taken at weekly intervals for evaluation of vaccine virusreplication and for EIA-specific immune responses. At 6 months postvaccination, all 16 vaccinated horses and 2 non-vaccinated controlhorses were challenged with the multiple low dose EIA equine challengewith EIAV_(PV) pathogenic virus stock as described previously. Themultiple low dose challenge involved inoculating each horse three timeswith 10 median horse infective doses (MHID) at two-day intervals. Thehorses were monitored for clinical signs of EIA, for seroconversion incommercial diagnostic assays for p26 and for infection with thechallenge virus using RT-PCR for about 3 months post challenge as inEXAMPLE 3. Table 3 summarizes the results of this study.

Seven of eight (88%) of the EIAV_(UK)ΔDUΔS2 vaccinated horses remainedasymptomatic post challenge, while six of eight (75%) of theEIAV_(PR)ΔS2 vaccinates were protected from disease post challenge.These clinical data indicate that the vaccines were effective inpreventing disease post challenge exposure to a pathogenic EIAV_(PV).However, these vaccines were not as effective as the vaccine tested inExample 3. It is proposed that the reduced protection results from theseconstructs either being prepared from an avirulent clone of EIA(EIAV_(PR)) or a double deletion mutant of the virulent parent clone(EIAV_(UK)ΔDUΔS2). It is proposed that addition of an adjuvant to thevaccines of this example would improve their immunogenicity (ability toprotect horses from disease) and produce a vaccine that is moreprotective for disease caused by EIA virus.

Surprisingly, not all of the vaccinated horses seroconverted to p26 asmeasured by testing for positive antibody status using the Coggins Test.This indicates that a normal p26 assay could be run on vaccinatedhorses. In order to use this vaccine for commercial purposes, anyvaccinated equines that were found to be Coggins Test positive could beconfirmed with a test for antibodies for the S2 expression product. IfS2 antibodies were present, it would be confirmed that the horses hadbeen infected with a field strain of EIAV (wild-type) and not the EIAVvaccine of the present invention.

It is apparent that a vaccine composition for effectively and safelyimmunizing equines from disease caused by EIAV can be produced and thatvaccinated equines can be differentiated from infected equines using thestandard Coggins test for antibodies to p26 in addition to a test forantibodies to S2 protein or detection of a gene sequence associated withthe S2 gene. Antibodies to both proteins as well as the S2 gene sequenceare absent in vaccinated and uninfected equines but present in infectedequines. Additionally, the absence of antibodies to the DU proteinand/or the DU gene sequence can serve as a differential diagnostic testfor equids vaccinated with the EIAV_(UK)ΔDUΔS2.

It is expected that the attenuated vaccines described in this examplewere more attenuated than desired. In order to increase theirimmunogenicity (ability to protect from disease and infection) anadjuvant can be added to the attenuated vaccine or the attenuatedviruses can be inactivated as described previously, adjuvanted andadministered as repeat doses (2 to 3) for the vaccination series. It isexpected that such a modification would protect completely from diseaseand infection.

TABLE 3 Summary of Attenuated EIAV Vaccine Trial P26 Febrile RNAEIAV_(PV) ANTIBODY Group Horse Episode >10⁵ Positive PositiveEIAV_(PR)ΔS2 811 X X X 9705 X X 9704 X 9717 X X 9615 X X X X 9613 X X X9716 X X 9712 X X EIAV_(UK)ΔDUΔS2 9708 X X 9706 X X 673 X X X 677 X 9711X 666 X X X X 711 699 Control 9714 X X X X 9720 X X X X

EXAMPLE 6

In order to determine whether a vaccine comprising only a DUgene-mutated EIAV would be safe and effective in equines, a DUgene-mutated EIAV construct was prepared and tested in a horsevaccination/challenge model for EIAV as described in Examples 3 and 4.The DU coding region of EIAV is located within the pol open readingframe, positioned between the RT and integrase (IN) genes (See FIG. 5).It specifically codes for a dUTPase, an enzyme to convert dUTP todUMP+pp₁ The predicted amino acid sequence of the EIAV DU protein showsa high degree of homology to the dUTPases of other nonprimatelentiviruses and to the human, yeast and E. coli enzymes as well. Fiveconserved amino acid motifs present in all known dUTPase proteins havebeen recognized and at least one of these motifs has been suggested tobe functionally important. Motif 3 contains a highly conserved tyrosineresidue, which has been suggested to be involved in catalysis. Toconstruct an EIAV mutant that would be deficient in dUTPase activity, aStyI restriction fragment containing 80% of the DU coding sequence,including four of the five conserved amino acid motifs, was deleted fromthe provirus clone EIAV_(PR). The deletion left intact the pol openreading frame and both protease-processing sites present on either sideof the DU gene. More specifically, to construct the EIAV_(PR)ΔDU that isdeficient in dUTPase activity, a 330 bp restriction fragment from aKpnI-PstI pol subclone of the proviral clone EIAV_(PR) was deleted. Thisdeleted segment was then subcloned back into a full-length provirusbackbone as an SstI-NcoI fragment to create the mutant provirus cloneEIAV_(PR)ΔDU (see FIG. 5). FIG. 5 shows the genomic organization of EIAVand the location of the DU gene. The position of the two StyI sites usedto create the deletion are also shown. The stippled bar represents theapproximate positions of five conserved amino acid motifs present in allknown dUTPase genes. Nucleotide and amino acid sequences of DU flankingthe two StyI sites are shown at the bottom. The leucine residue is thefirst amino acid of a true DU protein. A pol subclone containing the DUgene was digested with StyI, and the resulting 5′ termini were filled inwith T4 DNApolymerase and ligated to generate the sequence shown by thearrow. The deleted ˜gene was then inserted back into a full-lengthproviral clone.

The mutant produced as described, was tested for its ability toreplicate in vitro, a requirement for large-scale vaccine production.FEK cells and the ED cell line were transfected with the EIAV_(PR)ΔDU asdescribed previously in Example 2. It was determined that the RTactivity was equal to that of wild-type EIAV_(UK). However, when equinemacrophage cultures were transfected with this construct at amultiplicity of infection (MOI) of 0.01, very little replication (asmeasured by RT activity) was noted. This suggests that such a constructwould replicate poorly if at all in horses. The tissue culture grownproviral construct was confirmed to be EIAV_(PR)ΔDU by RT-PCR. Theseexperiments determined that EIAV_(PR)ΔDU could be produced in vitro inlarge scale in either FEK or ED cells.

In order to determine whether a vaccine could be prepared and whethersuch a vaccine would protect horses from disease and/or infection, theED cell line was transfected and a large quantity of EIAV_(PR)ΔDU wasproduced. In this study, the proviral construct was inactivated byaddition of 0.1% formalin and adjuvanted with a polymer-based adjuvant,specifically with a Carbopol-based adjuvant designated HAVLOGEN®. Twovaccines were formulated. One contained 50 μg/dose (1.0 mL) while thesecond contained 10 μg/dose. Each of three horses received 3 doses of 50μg/dose vaccine and each of three horses received 3 doses of 10 μg/dosevaccine. The interval between vaccinations was one month. Threeadditional horses were left unvaccinated and served as negativecontrols. Nine weeks post final vaccination, all horses were challengedwith a multiple low dose challenge using EIAV_(PV), a heterologousstrain. This constituted administering 10 MHIDs three times over a 7 dayperiod (days 0, 2 and 5). Horses were monitored for temperature,platelet count, plasma viremia and seroconversion for 7 weeks postchallenge. Results of this vaccination/challenge study are shown inTable 4.

TABLE 4 Summary of Results of the Vaccination/Challenge Study using aninactivated, Adjuvanted DU gene-mutated EIAV Vaccine Febrile RNAEIAV_(UK) P26 ANTIBODY Group Horse Episode >10⁵ Positive PositiveEIAV_(PR)ΔDU 710 None Neg Neg X 50 μg/dose 682 None Neg Neg X 95-03 NoneNeg Neg X EIAV_(PR)ΔDU 787 X X X X 10 μg/dose 785 X X X X 724 None NegNeg Neg Controls 96-08 X X X X 827 X X X X 746 X X X X

It is noted from Table 5 that all three horses receiving a 50 μg/dose ofinactivated, adjuvanted vaccine were protected from both disease andinfection. These horses demonstrated no clinical signs of disease anddid not demonstrate the presence of challenge virus (viremia) asmeasured by RT-PCR. Even a dose of only 10 μg was able to protect 1 of 3horses from both disease and infection. All control horses demonstratedboth disease and infection typical of full-blown EIA. This is anextraordinary result, especially since the challenge virus that wasadministered was heterologous, not homologous to the vaccine constructs.These data prove that the teachings of the present invention can be usedto prepare a completely protective vaccine. It also proves thatinactivation and adjuvanting do not decrease the immunogenicity of theEIAV vaccines of the present invention.

Although the invention has been described in detail in the foregoing,for the purpose of illustration it is to be understood that such detailis solely for that purpose and that variations can be made therein bythose skilled in the art without departing from the spirit and scope ofthe invention except as it may be limited by the claims.

1. A method of differentiating a vaccinated mammal from a non-vaccinatedmammal, said method comprising: a. obtaining a sample from a testmammal; and b. analyzing said sample for the presence or a geneexpression product normally produced by wild-type EIAV but not producedby an EIAV construct used for vaccinating said test mammal.
 2. Themethod of claim 1 wherein the step of analyzing said sample is performedwith the use of gene probes.
 3. The method of claim 1 wherein the stepof analyzing comprises testing said sample for the presence of aspecific antibody selected from the group consisting of a DU antibody, aS2 antibody, and both the DU antibody and the S2 antibody.
 4. The methodof claim 1 wherein the construct contains a non-functional gene selectedfrom the group of an S2 gene and a DU gene.
 5. The method of claim 1wherein the step of analyzing is performed with an assay selected fromthe group consisting of an ELISA, an antibody-detecting assay, and a PCRbased assay.
 6. The method of claim 2 wherein the EIAV construct furthercomprises a gene for another disease.
 7. Thc method of claim 1 whereinthe virus construct lacks the ability to express a mutated gene proteinfrom the mutated gene sequence in vivo.